Fig. 6: AAV-VEGF-C induced dLV expansion in APdE9 mice improves CSF outflow into cLNs and into blood circulation.
Comparison of littermate AAV-Mock and AAV-VC-treated i.c.m. (male) and i.c.v. (female) injected WT and APdE9 mice at 9 months of age. Rho, rhodamin; vp, viral particle; VC, vascular endothelial growth factor C (VEGF-C). a, Schedule indicating AAV administration and experimental analysis time points. b, Simplified schematic illustration of dLVs (green) attached to the basal and dorsal cranium and spinal canal after removal of the brain and spinal cord. c–f, Comparison of LYVE1 (white) staining in dorsal skull of i.c.m. (n = 4, 3, 3 and 3) (c,e) and i.c.v. (n = 3, 3, 3 and 3) (d,f) injected mice. Pineal gland was excised in (c,d) to visualize all dLVs. g–h, Experimental schedules for IgG–RPE and Rho-dextran drainage analysis into blood (g) and lymphatic system (g,h). i–l, Comparison of tracer signal in dcLNs at 180 min (n = 22, 23, 11 and 14) (i,j) and 30 min (n = 6, 5, 5 and 6) (k,l) after i.c.m. injection. m–p, Comparison of tracer signal in scLNs at 180 min (n = 20,22,8,13) (m,n) and 30 min (n = 6, 5, 5 and 7) (o,p) after i.c.m injection. q, Kinetic analysis of IgG–RPE tracer in systemic blood (saphenous vein) at 30, 60, 90, 120 and 180 min after i.c.m. injection (n = 21, 19, 12 and 15), visualized in three different ways. Data shown are representative of at least two independent experiments using littermate mice. Data points shown in graphs represent individual mice. Maximum one LN per side per mouse was used in quantification. IgG-RPE LN values represent an average of both sides. LN and blood tracer signal values were normalized to the average of WT-Ctrl group of every experimental set at 3 h (IgG–RPE) and 30 min (Rho-dextran) time point. P values were calculated using two-way ANOVA (e,f,j,l,n,p) and three-way repeated measures mixed-effects model with Tukey’s post hoc test for multiple comparison (q). Data are presented as mean ± s.e.m. Scale bars, 400 µm (c–d,i,k) and 1 mm (m,o).
